Abstract

We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25C is determined to be equal to (4.09 ± 0.07)x10^-6cm²/s, in good agreement with the value of 4.04x10^-6cm²/s as measured by 2fFCS.

© 2008 Optical Society of America

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History
Original Manuscript: April 10, 2008
Revised Manuscript: July 18, 2008
Manuscript Accepted: August 5, 2008
Published: September 3, 2008

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Author Affiliations

You Korlann, Thomas Dertinger, Xavier Michalet, Shimon Weiss

University of California, Los Angeles

Jörg Enderlein

Eberhard Karls University Tübingen

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